Differential bacterial gene expression during experimental pneumococcal endophthalmitis.

Streptococcus pneumoniae (pneumococcus) is a potential cause of bacterial endophthalmitis in humans that can result in ocular morbidity. We sought to identify pneumococcal genes that are differentially expressed during growth in the vitreous humor of the eye in an experimental endophthalmitis model. Microarray analysis was used to identify genes that were differentially expressed when pneumococci replicated in the vitreous of rabbit eyes as compared with bacteria grown in vitro in Todd Hewitt medium. Array results were verified by quantitative real-time PCR analysis of representative genes. Select genes potentially playing a role in virulence during endophthalmitis were deleted, and mutants were tested for reduced eye pathogenesis and altered adhesion to host cells. Array analysis identified 134 genes that were differentially expressed during endophthalmitis; 112 genes demonstrated increased expression during growth in the eye whereas 22 were downregulated. Real-time analysis verified increased expression of neuraminidase A (NanA; SP1693), neuraminidase B (NanB; SP1687) and serine protease (SP1954), and decreased expression of RlrA (SP0461) and choline transporter (SP1861). Mutation of NanA and NanB had no major effect on pathogenesis. Loss of SP1954 led to increased adherence to host cells. S. pneumoniae enhances and represses the expression of a variety of genes during endophthalmitis. While some of these genes reflect changes in metabolic requirements, some appear to play a role in immune evasion and pathogenesis in the eye.

The cholesterol-dependent cytolysin pneumolysin from Streptococcus pneumoniae binds to lipid raft microdomains in human corneal epithelial cells.

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both Ply(WT) and the mutant Ply molecules (Ply(A370G), Ply(A370E), Ply(A406G), Ply(A406E), Ply(W433G), Ply(W433E), Ply(W433F), Ply(L460G), and Ply(L460E)) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, Ply(WT) localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (Ply(A370G)) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (Ply(A370E), Ply(W433G), Ply(W433E), and Ply(L460E)) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except Ply(A370G), exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.

Passive immunization with Pneumovax® 23 and pneumolysin in combination with vancomycin for pneumococcal endophthalmitis.

BACKGROUND: Capsule and pneumolysin (PLY) are two major virulence factors of Streptococcus pneumoniae. S. pneumoniae is one of the leading causes of bacterial endophthalmitis. The aim of this study is to determine whether passive immunization with the 23-valent pneumococcal polysaccharide vaccine (Pneumovax® 23; PPSV23) or PLY protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were passively immunized with antiserum to PLY, PPSV23, a mixture of PPSV23/PLY, or PBS (mock). Vitreous was infected with a clinical strain of S. pneumoniae. In a separate group of experiments, vancomycin was injected 4 hours post-infection (PI) for each passively immunized group. Severity of infection, bacterial recovery, myeloperoxidase (MPO) activity and percent loss of retinal function were determined. RESULTS: Passive immunization with each antiserum significantly lowered clinical severity compared to mock immunization (PPSV23 = 9.19, PPSV23/PLY = 10.45, PLY = 8.71, Mock = 16.83; P = 0.0467). A significantly higher bacterial load was recovered from the vitreous of PLY passively immunized rabbits 24 hours PI (7.87 log10 CFU) compared to controls (7.10 log10 CFU; P = 0.0134). Retinas from immunized rabbits were more intact. Vitreous of PLY (2.88 MPO untis/mL) and PPSV23/PLY (2.17) passively immunized rabbits had less MPO activity compared to controls (5.64; P = 0.0480), and both passive immunizations (PLY = 31.34% loss of retinal function, PPSV23/PLY = 27.44%) helped to significantly preserve retinal function compared to controls (64.58%; P = 0.0323). When vancomycin was administered 4 hours PI, all eyes were sterile at 24 hours PI. A significantly lower clinical severity was observed for rabbits administered the combination immunization (5.29) or PPSV23 (5.29) with vancomycin treatment compared to controls (17.68; P = 0.0469). CONCLUSIONS: Passive immunization with antisera to these antigens is effective in reducing clinical severity of pneumococcal endophthalmitis in rabbits. Addition of vancomycin to immunization is effective at eliminating the bacteria.

Active Immunization with Pneumolysin versus 23-Valent Polysaccharide Vaccine for Streptococcus pneumoniae Keratitis.

PURPOSE: The purpose of this study was to determine whether active immunization against pneumolysin (PLY), or polysaccharide capsule, protects against the corneal damage associated with Streptococcus pneumoniae keratitis. METHODS: New Zealand White rabbits were actively immunized with Freund's adjuvant mixed with pneumolysin toxoid (ψPLY), Pneumovax 23 (PPSV23; Merck, Whitehouse Station, NJ), or phosphate-buffered saline (PBS), before corneal infection with 105 colony-forming units (CFU) of S. pneumoniae. Serotype-specific rabbit polyclonal antisera or mock antisera were passively administered to rabbits before either intravenous infection with 10¹¹ CFU S. pneumoniae or corneal infection with 105 CFU of S. pneumoniae. RESULTS: After active immunization, clinical scores of corneas of the rabbits immunized with ψPLY and Freund's adjuvant were significantly lower than scores of the rabbits that were mock immunized with PBS and Freund's adjuvant or with PPSV23 and Freund's adjuvant at 48 hours after infection (P ≤ 0.0010), whereas rabbits immunized with PPSV23 and Freund's adjuvant failed to show differences in clinical scores compared with those in mock-immunized rabbits (P = 1.00) at 24 and 48 hours after infection. Antisera from rabbits actively immunized with PPSV23 and Freund's adjuvant were nonopsonizing. Bacterial loads recovered from infected corneas were higher for the ψPLY- and PPSV23-immunized rabbits after infection with WU2, when compared with the mock-immunized rabbits (P ≤ 0.007). Conversely, after infection with K1443, the ψPLY-immunized rabbits had lower bacterial loads than the control rabbits (P = 0.0008). Quantitation of IgG, IgA, and IgM in the sera of ψPLY-immunized rabbits showed high concentrations of PLY-specific IgG. Furthermore, anti-PLY IgG purified from ψPLY-immunized rabbits neutralized the cytolytic effects of PLY on human corneal epithelial cells. Passive administration of serotype-specific antisera capable of opsonizing and killing S. pneumoniae protected against pneumococcal bacteremia (P ≤ 0.05), but not against keratitis (P ≥ 0.476). CONCLUSIONS: Active immunization with pneumococcal capsular polysaccharide and Freund's adjuvant fails to produce opsonizing antibodies, and passive administration of serotype specific opsonizing antibodies offers no protection against pneumococcal keratitis in the rabbit, whereas active immunization with the conserved protein virulence factor PLY and Freund's adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.

Comparison of besifloxacin, gatifloxacin, and moxifloxacin against strains of pseudomonas aeruginosa with different quinolone susceptibility patterns in a rabbit model of keratitis.

PURPOSE: Determine the effectiveness of topical besifloxacin, gatifloxacin, and moxifloxacin in treating keratitis caused by 2 strains of Pseudomonas aeruginosa with different quinolone susceptibility profiles. METHODS: Minimal inhibitory concentrations (MICs) were determined for each fluoroquinolone. Sequence analysis was performed on the quinolone resistance determining regions of the ciprofloxacin/levofloxacin-resistant strain. Rabbit corneas were injected with 10 colony-forming units (CFU). After 16 hours, phosphate-buffered saline, besifloxacin (6 mg/mL), gatifloxacin (3 mg/mL), or moxifloxacin (5 mg/mL) was applied topically every 15 minutes for 5 doses, then every 30 minutes for 14 doses. Eyes were examined pre- and posttreatment. Corneas were harvested for bacterial quantitation. RESULTS: MICs against the fully susceptible strain were 0.5, 0.25, and 0.5 µg/mL for besifloxacin, gatifloxacin, and moxifloxacin, respectively. The MICs against the ciprofloxacin/levofloxacin-resistant strain were 2, 16, and 32 µg/mL for besifloxacin, gatifloxacin, and moxifloxacin, respectively. Sequence analysis revealed amino acid mutations in all 4 fluoroquinolone target genes. None of the treatments had an effect on clinical severity of eyes infected with the fully susceptible strain (P > 0.05); however, all were effective at significantly reducing the bacterial CFU in the corneas (P < 0.05). For the ciprofloxacin/levofloxacin-resistant strain, clinical scores of besifloxacin-treated eyes were significantly lower than moxifloxacin-treated eyes (P < 0.037). The quantities of ciprofloxacin/levofloxacin-resistant bacteria recovered from corneas of all treatment groups were significantly lower than those recovered from phosphate-buffered saline-treated corneas (P < 0.05). Besifloxacin-treated eyes had significantly lower CFU recovered as compared with that of gatifloxacin- and moxifloxacin-treated eyes (P < 0.05). CONCLUSIONS: These results support clinical investigation of the effectiveness of besifloxacin in treating Pseudomonas keratitis.

The Streptococcus pneumoniae capsule is required for full virulence in pneumococcal endophthalmitis.

PURPOSE: To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. METHODS: An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. RESULTS: Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1ß, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. CONCLUSIONS: Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Pathogenesis of A Clinical Ocular Strain of Streptococcus pneumoniae and the Interaction of Pneumolysin with Corneal Cells.

Streptococcus pneumoniae is an important cause of bacterial keratitis, an infectious disease of the cornea. This study aimed to determine the importance of pneumolysin (PLY), a pneumococcal virulence factor, in keratitis using a clinical keratitis isolate (K1263) and its isogenic mutant deficient in PLY (K1263ΔPLY) and determine the effect of these strains on primary rabbit corneal epithelial (RCE) cells. Each strain was injected into the corneal stromas of rabbits, clinical examinations were performed, and the recovered bacterial loads were determined. Bacterial extracts were exposed to RCE cells, and morphology and viability were assessed. The mutant strain deficient in PLY, K1263ΔPLY, caused significantly lower ocular disease scores than the parent strain (K1263), although a higher bacterial load was recovered from corneas infected with the mutant strain. Histological examination showed increased inflammatory cells in the anterior chamber and increased edema in eyes infected with the parent strain. RCE cells exposed to the parent strain had significantly decreased cell viability and showed increased evidence of cellular damage. This study confirms that in a strain that can cause clinical keratitis, PLY is a significant cause of the damage associated with pneumococcal keratitis. It also shows for the first time that the results from an in vitro model using RCE cells correlates with in vivo results thereby establishing a less invasive way to study the mechanisms of pneumococcal keratitis.

Moxifloxacin and cholesterol combined treatment of pneumococcal keratitis.

PURPOSE: Compare the efficacy of treatment of pneumococcal keratitis with cholesterol, moxifloxacin, or a mixture of the two (moxifloxacin/cholesterol). MATERIALS AND METHODS: New Zealand white rabbits were injected intrastromally with 10(6) colony-forming units (CFU) of a clinical keratitis strain of Streptococcus pneumoniae. Eyes were examined before and after treatment of topical drops every 2 hr from 25 to 47 hr post-infection (PI). Corneas were harvested to quantitate bacterial CFU, and myeloperoxidase (MPO) activity was measured at 48 hr PI. Eyes were extracted for histology. Minimal inhibitory concentrations (MICs) were determined for each compound. RESULTS: Eyes treated with moxifloxacin/cholesterol had a significantly lower mean slit lamp examination (SLE) score than eyes treated with phosphate-buffered saline (PBS), moxifloxacin alone, or cholesterol alone (P ≤ 0.02). A significantly lower log(10) CFU was recovered from corneas treated with moxifloxacin/cholesterol and moxifloxacin alone as compared to corneas of eyes treated with PBS or cholesterol alone (P < 0.01). At 48 hr PI, significantly lower MPO activity was quantitated from eyes treated with moxifloxacin/cholesterol as compared to eyes treated with cholesterol or moxifloxacin alone (P ≤ 0.046). Eyes treated with moxifloxacin/cholesterol had fewer immune cells and less corneal destruction than eyes from all other treatment groups. The MIC for moxifloxacin alone was 0.125 µg/mL, and cholesterol alone was unable to inhibit growth at any of the concentrations tested. The MIC for moxifloxacin when combined with 1% cholesterol was 0.0625 µg/mL. CONCLUSIONS: Treatment with a mixture of moxifloxacin and cholesterol significantly lowers the severity of infection caused by pneumococcal keratitis as compared to treatment with moxifloxacin alone, cholesterol alone, or PBS. This treatment mixture eradicates the bacteria in the cornea, unlike treatment with PBS or cholesterol alone. Using cholesterol with moxifloxacin as a treatment for bacterial keratitis could help lower the clinical severity of the infection.

Immunization with pneumolysin protects against both retinal and global damage caused by Streptococcus pneumoniae endophthalmitis.

PURPOSE: To determine whether immunization with pneumolysin (PLY) protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were immunized with a mutant form of PLY that retains only 1% of its cytolytic activity until serum IgG titers were ≥51,200. For a negative control, rabbits were immunized with phosphate-buffered saline (mock). Each vitreous was injected with 10(2) colony-forming units of a clinical endophthalmitis isolate of Streptococcus pneumoniae. Severity of endophthalmitis was graded by slit lamp examination at 24 and 48 h postinfection (PI). Serial dilutions of vitreous were plated for bacterial colony-forming units quantitation, eyes were extracted for histology, and a whole blood survival assay was performed. RESULTS: Immunized rabbits had a significantly lower mean slit lamp examination score at 24 and 48 h PI when compared to mock immunized rabbits (P ≤ 0.002). There was not a significant difference in bacterial load in the vitreous at 24 or 48 h PI. Histological sections showed that retinas of mock immunized rabbits appeared to be destroyed, whereas those of PLY immunized rabbits remained largely intact. Damage spread to the aqueous humor, stroma, and conjunctiva of mock immunized rabbits by 48 h PI. Minimal damage was observed in the vitreous of PLY immunized rabbits and did not spread to other parts of the eye. Whole blood from immunized rabbits inhibited the growth of bacteria better than whole blood from mock immunized rabbits. CONCLUSION: Immunization with PLY helps protect the eye from damage caused by pneumococcal endophthalmitis.

Assessment of Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase activity increases in nonencapsulated pneumococci following conjunctival infection.

PURPOSE: The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection. METHODS: A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth. RESULTS: There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection. CONCLUSIONS: Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

Comparative efficacy of besifloxacin and other fluoroquinolones in a prophylaxis model of penicillin-resistant Streptococcus pneumoniae rabbit endophthalmitis.

PURPOSE: To study the efficacy of topical administration of gatifloxacin (0.3%), moxifloxacin (0.5%) ophthalmic solutions, and besifloxacin (0.6%) ophthalmic suspension as prophylaxis and treatment of pneumococcal endophthalmitis. METHODS: Four groups of New Zealand white rabbits were topically treated with gatifloxacin, moxifloxacin, besifloxacin, and phosphate-buffered saline (PBS) at the following time points: 60, 45, 30, and 15 min before infection, immediately after infection, and then 6, 12, 18, and 24 h postinfection. Penicillin-resistant Streptococcus pneumoniae (PRSP; 10(6) colony-forming unit [CFU] in 50 microL) was injected into the aqueous humor of each eye. The clinical severity of the eyes was assessed by 2 masked observers 24 h postinfection. Aqueous and vitreous samples were collected, diluted, and plated to determine recovered CFU. RESULTS: Fluoroquinolone-treated eyes had significantly lower clinical scores and bacteria recovered from the aqueous humor than the PBS-treated eyes. There was no difference, however, among the fluoroquinolone-treated groups. In contrast, none of the fluoroquinolones reduced the number of bacteria recovered (CFU) from the vitreous humor. CONCLUSIONS: Besifloxacin is as effective as moxifloxacin and gatifloxacin in a rabbit model for topical prophylaxis and treatment of PRSP-induced endophthalmitis.

Efficacy of besifloxacin in an early treatment model of methicillin-resistant Staphylococcus aureus keratitis.

PURPOSE: To determine the effectiveness of topically applied besifloxacin, gatifloxacin, and moxifloxacin for the early treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS: Ten hours post-MRSA infection, rabbit eyes were treated topically with 19 doses of phosphate-buffered saline (PBS), besifloxacin, gatifloxacin, or moxifloxacin. Slit-lamp examinations were performed before and after the inoculation. Corneas were harvested for bacterial quantitation and minimal inhibitory concentrations (MICs) were determined. RESULTS: All 3 fluoroquinolones significantly lowered the clinical severity of the infection as compared to treatment with PBS (P < 0.05). However, the mean log(10) colony-forming unit (CFU) recovered from besifloxacin-treated corneas was significantly lower than all other treatment groups (P < 0.01). CFU recovered from corneas treated with moxifloxacin and PBS showed no significant difference (P = 0.12). Corneas treated with gatifloxacin had a significantly lower log(10) CFU recovered as compared to PBS-treated corneas (P < 0.01). The MICs for gatifloxacin and moxifloxacin were 8 microg/mL, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSIONS: All 3 fluoroquinolones significantly lowered the clinical severity of the infection. Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin, and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea.

Efficacy of besifloxacin in a rabbit model of methicillin-resistant Staphylococcus aureus keratitis.

PURPOSE: The purpose of this study was to determine the effectiveness of topically applied besifloxacin (0.6%), gatifloxacin (0.3%), and moxifloxacin (0.5%) for the late treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS: One hundred colony-forming units (CFUs) of bacteria were injected intrastromally into rabbit corneas. Sixteen hours after infection, one topical drop of phosphate-buffered saline, besifloxacin, gatifloxacin, or moxifloxacin was applied to each eye every 15 minutes for 5 doses and then every 30 minutes for 14 doses. Eyes were examined before and after treatment by slit lamp biomicroscopy. Corneas were harvested from treated and untreated rabbits for the quantitation of bacteria. Minimal inhibitory concentrations (MICs) were determined in vitro for each fluoroquinolone. RESULTS: None of the treatments had an effect on clinical severity (P > 0.05). Although there were no differences in clinical severity between any groups after treatment, the mean log10 CFU of MRSA recovered from besifloxacin-treated corneas (5.111 +/- 0.251) was significantly lower than the CFU recovered from corneas treated with phosphate-buffered saline (7.006 +/- 0.144), gatifloxacin (7.108 +/- 0.346), and moxifloxacin (7.473 +/- 0.144; P < 0.001). CFU recovered from gatifloxacin- and moxifloxacin-treated corneas were not significantly different from phosphate-buffered saline-treated corneas (P = 1.000). The MICs against the MRSA strain were 8 microg/mL for both gatifloxacin and moxifloxacin, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSION: Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea 16 hours after infection.

Development of a Streptococcus pneumoniae keratitis model in mice.

BACKGROUND: Streptococcus pneumoniae is a common cause of bacterial keratitis, and models to examine the ocular pathogenesis of this bacterium would aid in efforts to treat pneumococcal keratitis. The aim of this study was to establish a murine model of pneumococcal keratitis. METHODS: The corneas of A/J, BALB/c or C57BL/6 mice were scratched and topically infected with a clinical strain of S. pneumoniae. Slitlamp examination (SLE), enumeration of bacteria in the corneas and histology were performed. RESULTS: Bacteria were recovered from the eyes of A/J mice on postinfection (PI) days 1 [1.96 +/- 0.61 log(10) colony-forming units (CFU)] and 3 (1.41 +/- 0.71 log(10) CFU). SLE scores were significantly higher in the infected A/J mice as compared to the BALB/c or C57BL/6 mice on PI day 3 (p < 0.0001) and steadily increased over time, reaching a maximal value of 3.00 +/- 0.35 on PI day 10. Histopathology revealed stromal edema and the influx of polymorphonuclear leukocytes on PI days 7 and 10, and corneal disruption on PI day 7. CONCLUSIONS: S. pneumoniae keratitis was established in A/J mice, but not BALB/c or C57BL/6 mice.

A comparison of pneumolysin activity and concentration in vitro and in vivo in a rabbit endophthalmitis model.

The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis. Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Rabbit vitreous was injected with 10(2) colony-forming units (CFU) of 1 of 2 different strains with low hemolytic activity (n = 10 and 12 for strains 4 and 5, respectively) or 1 of 3 different strains with high hemolytic activity (n = 12 per strain). Pathogenesis of endophthalmitis infection was graded by slit lamp examination (SLE) at 24 hours post-infection. Bacteria were recovered from infected vitreous and quantitated. The SLE scores of eyes infected with strains having high hemolytic activity were significantly higher than the scores of those infected with strains having low hemolytic activity (P < 0.05). Pneumolysin concentration in vitro, however, did not correlate with hemolysis or severity of endophthalmitis. Bacterial concentrations from the vitreous infected with 4 of the strains were not significantly different (P > 0.05). These data suggest that pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. The protein concentration of pneumolysin, however, is not a reliable indicator of pneumolysin activity.

Cholesterol as treatment for pneumococcal keratitis: cholesterol-specific inhibition of pneumolysin in the cornea.

PURPOSE: The purpose of this study was to determine whether cholesterol, the host cell receptor for pneumolysin of Streptococcus pneumoniae, could effectively treat pneumococcal keratitis. METHODS: New Zealand White rabbits were intrastromally injected with 10(5) colony-forming units (CFUs) of S. pneumoniae D39. Corneas were treated with topical drops of 1% cholesterol every 2 hours beginning 25 hours after infection and were examined by slit lamp microscopy 24, 36, and 48 hours after infection. Rabbits were killed, and CFUs were recovered from the corneas after the final slit lamp examination (SLE). Minimal inhibitory concentration (MIC) assays of cholesterol against bacteria were performed. Specific inhibition of pneumolysin by cholesterol in the rabbit cornea was tested by intrastromal injection of pneumolysin with or without cholesterol and was compared with cholesterol inhibition of pneumolysin in vitro using hemolysis assays with rabbit erythrocytes. RESULTS: Corneas treated with cholesterol had significantly lower SLE scores 48 hours after infection than corneas treated with vehicle (P = 0.0015). Treated corneas also had significantly less log(10) CFUs than vehicle-treated corneas (P = 0.0006). Cholesterol at a 1% concentration was bactericidal to bacteria in vitro, and lower concentrations of cholesterol were partially inhibitory in a concentration-dependent manner. Cholesterol also specifically inhibited 1 mug pneumolysin in vivo and up to 50 ng pneumolysin in vitro. CONCLUSIONS: Topical cholesterol is an effective treatment for S. pneumoniae keratitis. Cholesterol not only inhibits pneumolysin, it is also bactericidal.